The invention relates to the whole-body external optical imaging of gene expression. Specifically, methods for whole-body external optical imaging of gene expression and methods for evaluating a candidate protocol or drug for treating diseases or disorders using a fluorophore operatively linked to the promoter of a gene and external optical imaging are provided herein. Methods to screen for substances or genes that regulate target promoters are also provided.
Whole-body imaging technology has been used to monitor xe2x80x9ctracer moleculesxe2x80x9d in the intact body. For example, Brenner et al. studied the diagnostic value of iodine-123-2-hydroxy-3-iodo-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl] benzamide (IBZM) whole-body imaging in comparison to thallium-201 scintigraphy in patients with metastatic malignant melanoma (Brenner et al., Eur. J. Nucl. Med., 26(12):1567-71 (1999)). Benard et al. conducted clinical evaluation of processing techniques for attenuation correction with 137Cs in whole-body PET imaging (Benard et al., J. Nucl. Med., 40(8):1257-63 (1999)). Jerusalem et al. showed that whole-body positron emission tomography using 18F-fluorodeoxyglucose for posttreatment evaluation in Hodgkin""s disease and non-Hodgkin""s lymphoma has higher diagnostic and prognostic value than classical computed tomography scan imaging (Jerusalem et al., Blood, 94(2):429-33 (1999)). Eustace et al. discussed practical issues, clinical applications, and future directions of whole-body MR imaging (Eustace et al., Magn. Reson. Imaging Clin. (N. Am), 7(2):209-36 (1999)). Engelson et al. studied fat distribution in HIV-infected patients reporting truncal enlargement quantified by whole-body magnetic resonance imaging (Engelson et al., Am. J. Clin. Nutr., 69(6 :1162-9 (1999)). Valk et al. used whole-body positron emission tomography (PET) imaging with [18F]fluorodeoxyglucose in management of recurrent colorectal cancer (Valk et al., Arch. Surg., 134(5):503-11 (1999)). Saunders et al. evaluated fluorine-18-fluorodeoxyglucose whole body positron emission tomography imaging in the staging of lung cancer (Saunders et al., Ann. Thorac. Surg., 67(3):790-7 (1999)).
U.S. Pat. No. 5,650,135 discloses a noninvasive method for detecting the localization of an entity under study from within a mammalian subject, which method comprises: (a) administering to the subject a conjugate of the entity and a light-generating moiety or a transformed cell expressing the light-generating moiety; (b) after a period of time in which the conjugate or transformed cell can achieve localization in the subject, immobilizing the subject within the detection field of a photodetector device; (c) maintaining the subject in an immobilized condition, (d) during said maintaining, measuring photon emission from the light-generating moiety, localized in the subject, with the photodetector device until an image of photon emission can be constructed; and (e) detecting said image through an opaque tissue of said mammal. U.S. Pat. No. 5,650,135 also discloses a noninvasive method for detecting the level of an entity under study in a mammalian subject over time, which method comprises: (a) administering to the subject a conjugate of the entity and a light-generating moiety or a transformed cell expressing the light-generating moiety; (b) placing the subject within the detection field of a photodetector device; (c) maintaining the subject in the detection field of the device; (d) during said maintaining, measuring photon emission from the light-generating moiety, in the subject, with the photodetector device; and (e) repeating steps (b) through (d) at selected intervals, wherein said repeating is effective to detect changes in the level of the entity in the subject over time.
Recently, Yang et al. conducted whole-body optical imaging of green fluorescent protein-expressing tumors and metastases (Yang et al., Proc. Natl. Acad. Sci. (USA), 97(3): 1206-11 (2000)). Yang et al. have imaged, in real time, fluorescent tumors growing and metastasizing in live mice. The whole-body optical imaging system is external and noninvasive. It affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals. Yang et al. have established new human and rodent tumors that stably express very high levels of the Aequorea victoria green fluorescent protein (GFP) and transplanted these to appropriate animals. B16F0-GFP mouse melanoma cells were injected into the tail vein or portal vein of 6-week-old C57BL/6 and nude mice. Whole-body optical images showed metastatic lesions in the brain, liver, and bone of B 16F0-GFP that were used for real time, quantitative measurement of tumor growth in each of these organs. The AC3488-GFP human colon cancer was surgically implanted orthotopically into nude mice. Whole-body optical images showed, in real time, growth of the primary colon tumor and its metastatic lesions in the liver and skeleton. Imaging was with either a trans-illuminated epifluorescence microscope or a fluorescence light box and thermoelectrically cooled color charge-coupled device camera. The depth to which metastasis and micrometastasis could be imaged depended on their size. A 60-micrometer diameter tumor was detectable at a depth of 0.5 mm whereas a 1, 800-micrometer tumor could be visualized at 2.2-mm depth. The simple, noninvasive, and highly selective imaging of growing tumors, made possible by strong GFP fluorescence, enables the detailed imaging of tumor growth and metastasis formation. This should facilitate studies of modulators of cancer growth including inhibition by potential chemotherapeutic agents.
Methods for monitoring gene expression are known in the art (see generally, Ausubel et al. (Ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc.). However, whole-body external optical imaging of gene expression, which offers simple, noninvasive, highly selective, and real-time recording and analysis of gene expression in an intact multi-cellular organisms, e.g., animals, is not available currently. The present invention addresses this and other related needs in the art.
The invention provides for whole-body external optical imaging of gene expression and methods for evaluating a candidate protocol or drug for treating diseases or disorders. The method uses a fluorophore operatively linked to the promoter of a gene and external optical imaging. Methods to screen for substances or genes that regulate target promoters are also provided.
In a specific embodiment, a method to monitor the expression of a gene is provided, which method comprises: a) delivering to a multi-cellular organism a nucleic acid encoding a fluorophore operatively linked to the promoter of a gene whose expression is to be analyzed or delivering a cell containing said nucleic acid; and b) observing the presence, absence or intensity of the fluorescence generated by said fluorophore at various locations in said organism by whole-body external fluorescent optical imaging, whereby the expression of said gene is monitored.
In a preferred embodiment, a nucleic acid encoding a fluorophore operatively linked to the promoter of the gene is delivered directly to the organism. Also preferably, the nucleic acid encoding a fluorophore operatively linked to the promoter of the gene is in a viral vector such as a viral vector derived from adenovirus or a lentivirus.
In another preferred embodiment, a cell containing a nucleic acid encoding a fluorophore operatively linked to the promoter of the gene is delivered to the organism. More preferably, the cell is delivered to the organism via a surgical procedure such as direct implantation by surgical orthotopic implantation (SOI) at a desired site.
In still another preferred embodiment, the fluorophore operatively linked to the promoter of a gene is a humanized fluorophore. Also preferably, the fluorophore is a green fluorescent protein (GFP), a blue fluorescent protein (BFP) or a red fluorescent protein (RFP). More preferably, the GFP is the humanized hGFP-S65T.
In yet another preferred embodiment, the multi-cellular organism to be analyzed is a plant or an animal, including a transgenic animal. More preferably, the animal is a mammal. A human can also be analyzed by the present method.
In yet another preferred embodiment, the gene to be analyzed is expressed in a tissue or organ specific manner. More preferably, the gene is expressed in connective, epithelium, muscle or nerve tissue. Also more preferably, the gene is expressed in an internal animal organ such as brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, gland, internal blood vessels, etc. Yet more preferably, the gene to be analyzed is a tumor or cancer associated gene such as an oncogene or a tumor suppressor gene.
In yet another preferred embodiment, the expression of more than one gene is monitored simultaneously.
In another specific embodiment, a method to evaluate a candidate protocol or drug for treating a disease or disorder is provided, which method comprises: a) administering said protocol or drug to a non-human mammalian subject which expresses a fluorophore under the direction of a promoter of a gene associated with a disease or disorder, and determining the expression of said promoter via observing the presence, absence or intensity of the fluorescence generated by said fluorophore at various locations in said mammalian subject by whole-body external fluorescent optical imaging; b) determining the expression of said promoter, via observing the presence, absence or intensity of the fluorescence generated by said fluorophore at various locations in said mammalian subject by whole-body external fluorescent optical imaging, in a control non-human mammalian subject which expresses said fluorophore under the direction of said promoter of said gene; and c) comparing the expression of said promoter determined in steps a) and b), wherein the expression determined in step a) is different from that in step b) identifies said protocol or drug as effective in treating the disease or disorder.
If overexpression of the gene is associated with the disease or disorder, the expression determined in step a) is lower than that in step b) when said protocol or drug is effective in treating the disease or disorder.
If underexpression of the gene is associated with the disease or disorder, the expression determined in step a) is higher than that in step b) when said protocol or drug is effective in treating the infection.
Preferably, the disease or disorder is a cancer, an immune system disease or disorder, a metabolism disease or disorder, a muscle and bone disease or disorder, a nervous system disease or disorder, a signal disease or disorder, or a transporter disease or disorder.
Preferably, the non-human mammalian subject which expresses a fluorophore under the direction of a promoter of the gene is produced by delivering a nucleic acid encoding the fluorophore operatively linked to the promoter of the gene, or a cell containing the nucleic acid, to the non-human mammalian subject. Alternatively, the non-human mammalian subject used in the screen is a transgenic animal.
The non-human mammalian subject used in the screening is preferably a well established laboratory animal such as a mice, a rabbit or a non-human primate.
The fluorophore used in the screening is preferably a green fluorescent protein (GFP), a blue fluorescent protein (BFP) or a red fluorescent protein (RFP).
More than one candidate protocol or candidate drug is preferably screened for simultaneously.
If the non-human mammalian subject expresses a fluorophore under the direction of a promoter of an infectious organism, the expression determined in step a) is lower than that in step b) when said protocol or drug is effective in treating infection caused by the infectious organism.
The non-human mammalian subject used in the screening is preferably an infectious disease animal model.
The infectious organism screened against is preferably a fungus such as a yeast, a bacterium such as an eubacteria or an archaebacteria, or a virus such as a Class I virus, a Class II virus, a Class III virus, a Class IV virus, a Class V virus or a Class VI virus.
If the infection is caused by a bacterium, the candidate drug to be screened is preferably an antibiotic.
In still another specific embodiment, a method to screen for a modulator of the expression of a gene in a non-human multi-cellular organism is provided, which method comprises: a) administering a test substance to a non-human multi-cellular organism which expresses a fluorophore under the direction of a promoter of a gene, and determining the expression of said promoter via observing the presence, absence or intensity of the fluorescence generated by said fluorophore at various locations in said multi-cellular organism by whole-body external fluorescent optical imaging; b) determining the expression of said promoter, via observing the presence, absence or intensity of the fluorescence generated by said fluorophore at various locations by whole-body external fluorescent optical imaging, in a control multi-cellular organism which expresses said fluorophore under the direction of said promoter of said gene; and c) comparing the expression of said promoter determined in steps a) and b), wherein the expression determined in step a) is different from that in step b) identifies said test substance as a modulator of said gene expression. Preferably, the promoter is an endogenous promoter of the multi-cellular organism.
In yet another specific embodiment, a method to screen for a non-human multi-cellular organism that expresses a gene at an altered level is provided, which method comprises: a) administering a mutation-inducing agent or treatment to a non-human multi-cellular organism which expresses a fluorophore under the direction of a promoter of a gene, and determining the expression of said promoter via observing the presence, absence or intensity of the fluorescence generated by said fluorophore at various locations in said multi-cellular organism by whole-body external fluorescent optical imaging; b) determining the expression of said promoter, via observing the presence, absence or intensity of the fluorescence generated by said fluorophore at various locations by whole-body external fluorescent optical imaging in an untreated control multi-cellular organism which expresses said fluorophore under the direction of said promoter of said gene; and c) comparing the expression of said promoter determined in steps a) and b), wherein the expression determined in step a) is different from that in step b) identifies a multi-cellular organism that expresses said gene at the altered level. Preferably, the mutation-inducing agent or treatment causes a mutation in germ-line cells of the multi-cellular organism so that the desired mutation is stably-transferable to offspring of the multi-cellular organism.